Anti-V5, FLAG and GAPDH antibodies were purchased from Proteintech. Anti-α-Tubulin and anti-HA antibodies were purchased from Sigma. Anti-Sox2, Nanog and Oct4 antibodies were purchased from Abcam. The following antibodies were used: anti-FKBP9 (Invitrogen), anti-Calnexin (Santa Cruz), anti-Nestin (R&D Systems), anti-pmTOR (Invitrogen), anti-pP70S6K (Millipore), anti-pERK1/2 (Promega), anti-p65 (EPITOMICS), anti-ASK1 (Santa Cruz), anti-pASK1 (Santa Cruz), anti-pIRE1α (NOVUS). Other antibodies for immunoblotting were purchased from Cell Signaling Technology. Aggresome Detection Kit was purchased from Abcam. Thapsigargin (Tg) and tunicamycin (Tm) were purchased from Apexbio. Proteasome inhibitor MG132 and lysosomal inhibitors Bafalomycin A1 (Baf A1) and chloroquine (CQ) were obtained from Sigma. Drugs were dissolved and stored at − 20 °C or − 80 °C according to the instructions.
Aggresome detection kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Aggresome Detection Kit is a fluorescence-based assay designed to detect and visualize the formation of aggresomes, which are perinuclear inclusions composed of misfolded proteins and cellular waste. The kit provides reagents and protocols to label aggresomes and subcellular organelles, enabling researchers to study protein aggregation and cellular stress responses.
Automatically generated - may contain errors
Lab products found in correlation
Anti sox2, by Abcam (1 mentions)
Anti ask1, by Santa Cruz Biotechnology (1 mentions)
Anti p65, by Abcam (1 mentions)
Anti pire1α, by Novus Biologicals (1 mentions)
Anti nestin, by R&D Systems (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Anti v5, by Proteintech (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Nanog, by Abcam (1 mentions)
Anti calnexin, by Santa Cruz Biotechnology (1 mentions)
Mg132, by Merck Group (1 mentions)
Anti ha, by Merck Group (1 mentions)
Gapdh, by Proteintech (1 mentions)
Necrostatin 1 nec 1, by Selleck Chemicals (1 mentions)
N acetylcysteine nac, by Selleck Chemicals (1 mentions)
Cox 4, by Abcam (1 mentions)
Phospho drp1 ser616, by Cell Signaling Technology (1 mentions)
Mitosox detection kit, by Thermo Fisher Scientific (1 mentions)
Ad mrfp gfp lc3, by Beyotime (1 mentions)
Mdivi 1, by Selleck Chemicals (1 mentions)
6 protocols using aggresome detection kit
1
Antibody Validation and ER Stress Assays
2
Investigating Organelle-Specific Autophagy Pathways
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Antibodies against Drp1, p62, LC3, RIP1, RIP3, TSG101, and CD63 were purchased from Abcam (Cambridge, MA, USA). β-actin, tubulin, and COX IV, which were used as references for the total, cytoplasmic, and mitochondrial fractions, were also acquired from Abcam (Cambridge, MA, USA). Antibodies against Phospho-RIP3 (Thr231/Ser232) and Phospho-Drp1 (Ser616) were purchased from Cell Signaling Technology (Danvers, MA, USA). The Aggresome Detection Kit was acquired from Abcam (Cambridge, MA, USA). MitoTracker Deep Red, the ROS 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) kit, dihydroethidium (DHE) assay kit, and MitoSOX detection kit were purchased from Invitrogen (Carlsbad, CA, USA). Ad-mRFP-GFP-LC3 was supplied by Beyotime (Shanghai, China). Adenoviral vectors for Drp1 short hairpin RNA (shRNA) and Drp1 mutations (Drp1-S616D and Drp1-S616A) were generated by Genechem Technology (Shanghai, China). Related activators and inhibitors, including Mitochondrial division inhibitor 1 (Mdivi-1), N-acetylcysteine (NAC), and Necrostatin-1 (Nec-1), were obtained from Selleck (Shanghai, China). The Protein A/G Magnetic Beads IP Kit was purchased from Thermo Scientific (Waltham, MA, USA), and fetal bovine serum (FBS) and penicillin/streptomycin were procured by Invitrogen (Carlsbad, CA, USA). All other chemicals were supplied by Sigma unless otherwise specified.
3
Aggresome Detection in HeLa Cells
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WT or PACSIN1 KO HeLa cells were treated with 20 μM MG132 or 2.5 μg/mL puromycin for 6 h. Detergent soluble-insoluble proteins were fractionated using 1% triton-X 100 with PBS [62 (link)]. After centrifugation, the supernatant was used as the soluble fraction. The pellet was extracted with RIPA buffer included 7 M Urea and used as the insoluble fraction. Aggresome staining was performed by aggresome detection kit (abcam, ab139486) according to the manufacturer’s instruction. The number of aggresome dots normalized per cell were quantified by Fiji.
4
Detecting Amyloid Aggregates in Yeast
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In order to characterize the detected Hsp104-positive aggregates for the presence of amyloid structures, we employed the Aggresome detection kit (Abcam) according to the manufacturer’s instructions. Briefly, exponentially growing cells were subjected to starvation in 0.02% glucose for 90 min. The negative control cells were kept constatntly in 2% glucose. The positive control yeast strain, bearing the Sup35-GFP genomic fusion, were subjected to a 20-min heat shock at 37 °C in exponential growth stage. This was followed by washing the cells in 10 mM PBS and staining in the Aggresome detection kit in the presence of 8 mg/L DMSO to facilitate the entry of the dye into the yeast cells. Colocalization between Hsp104-GFP, as well as Sup35-GFP and red-stained foci (indicative of amyloid structure), was scored in at least 500 cells from three biological replicates, each in three technical replicates.
5
Aggresome Detection Protocol via Flow Cytometry
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The aggresome detection kit (ab139486) was from Abcam, Cambridge, UK. Cells were detached by trypsin/EDTA and centrifuged at 1.300 rpm for 5 min. Pellets were washed in 2 mL PBS, centrifuged (5 min, 1.300 rpm), and supernatants were discarded. Pellets were resuspended in 200 µL PBS and added dropwise to 2 mL of a 4% formaldehyde solution (37% formalin in 1 × assay buffer). The tubes were mixed carefully to complete the fixation step. After the cell suspensions were incubated for 30 min at room temperature, a centrifugation step at 800 × g for 15 min was done. The supernatants were removed, pellets resuspended in 2 mL PBS, and centrifuged at 800 × g for 15 min. To permeabilize the cells, 0.5% triton X-100 and 3 mM EDTA (pH = 8) in 1 × assay buffer was added by gentle mixing and incubation for 30 min on ice. Afterwards, cells were centrifugated at 800 × g for 15 min and washed with PBS. The cell suspensions were placed in a cell strainer and centrifuged to remove cellular debris. To detect aggregates, 500 µL of freshly prepared aggresome red detection reagent (1:5.000 in 1 × assay buffer) was added to the pellets and incubated for 30 min in the dark. Samples were measured with FACS Canto flow cytometer and analyzed with the BD FACSDiva™ Software.
6
Aggresome Staining and Immunoassay Protocol
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HEK293T and HeLa cells were seeded at approximately 1.75 × 105/well in a 24-well plate. Transfection was performed when cells were 60% confluent. Aggresome staining was performed according to the manufacturer’s manual (aggresome detection kit; Abcam; #ab139486). Briefly, transfected cells were fixed with 4% formaldehyde and permeabilized by 0.5% Triton X-100 before staining with the aggresome dye and DAPI. Positive control cells were treated with 10 µM MG132 for 24 h. For immunostaining, fixed and permeabilized cells were blocked in phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA) followed by anti-HA (#MMS-101R; Covance), anti-FLAG (#PA1-984B; Thermo Fisher Scientific), anti-Tom20 (#42406; Cell Signaling), anti-SdhA (#5839; Cell Signaling), anti-LC3 (#3868; Cell Signaling) antibodies for 1 h at 37°C. After washing with PBST, cells were then incubated with fluorescein-labeled secondary antibody for 45 min at 37°C. Stained cells were visualized using an Image Xpress microconfocal high-content imaging system (Molecular Devices).
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